Stem cells are undifferentiated cells that possess two hallmark properties; self-renewal and the ability to differentiate into one or more different cell lineages. The process of self-renewal involves the self-replication of a stem cell to allow for propagation and expansion, wherein the stem cell remains in an undifferentiated state. Progenitor cells are also undifferentiated cells that have the ability to differentiate into one or more cell lineages, but have limited or no ability to self renew. When maintained in culture, undifferentiated cells, such as stem or progenitor cells, can undergo spontaneous differentiation, thereby losing the desired, undifferentiated cell phenotype. Thus, culture methods that minimize spontaneous differentiation in order to maintain the undifferentiated stem or progenitor cell state are needed.
Keeping undifferentiated cells, such as, but not limited to, stem and/or progenitor cells in an undifferentiated state is critical to their use, e.g., in industry and medicine, since a major scientific and therapeutic usefulness of these cells lies in their ability to expand into homogenous populations that can further proliferate or differentiate into mature cells as needed, e.g., for scientific study or to repair damage to cells or tissues of a patient. Once they have spontaneously differentiated in cell culture, the cells are less proliferative and less able to differentiate into different types of cells as needed. A homogenous culture of undifferentiated stem cells is therefore a highly sought after but unrealized goal of research scientists and industry.
Current methods for culturing undifferentiated cells (e.g., various types of stem cells) attempt to minimize such spontaneous differentiation by delivering fibroblast growth factor 2 (FGF2) to the cell cultures daily, or, less frequently than every day, which is known as “feeding”. FGF2 has been shown to promote self-renewal of stem cells by inhibiting differentiation of the stem cell; however this inhibition is incomplete, and the stem cell cultures tend to gradually differentiate, thereby diminishing usefulness of the stem cell culture. Furthermore, stem cells, such as ES cells, typically need to be grown on mouse embryonic fibroblast (MEF) feeder cells. This is a cumbersome step that is desirable to remove.
For human embryonic stem cells (hESC), as well as other undifferentiated cell types, FGF2 is required for the maintenance of the undifferentiated state, and withdrawal of FGF2 from the culture conditions initiates differentiation. [See, Amit, M., et al. (2000) Dev Biol. 2, 271-78; Itskovitz-Eldor, J., et al. (2000) Mol. Med. 2, 88-95; Xu, C., et al. (2001) Nat. Biotechnol. 10, 971-74; Xu et al., 2005; Xu, R. H., et al. (2005) 3, 164-65; Ding, V., et al. (2006) Biotechnol Lett. 7, 491-95; Levenstein, M. E., et al. (2006) Stem Cells 3, 568-74; Ludwig, T. E., et al. (2006) Nat. Methods. 8, 637-46; Bendall, S. C., et al. Nature 448, 1015-21]. FGF2 is also required to maintain neural stem cells (NSC) and neural progenitor cells in an undifferentiated state. [See, Temple S. (1989). Nature 340:471-473.; Vescovi, A. L., et al. (1993) Neuron 5, 951-56; Kilpatrick, T. J., and Bartlett, P. F. (1995) J. Neurosci. 5, 3653-61; Temple, S., and Qian, X. (1995) Neuron 2, 249-52; Qian, X., et al. (1997) Neuron 1, 81-83; Ciccolini, F., and Svendsen, C. N. (1998) J. Neurosci. 19, 7869-80; Vaccarino F M, et al. (1999) Curr Top Dev Biol. 46 179-00; Raballo R, et al. J. Neurosci. 13, 5012-23.] In traditional methods for culturing NSCs, growth factors such as FGF2 are only replenished once every three days. However, NSCs cultured by these methods are reported to have high rates of spontaneous differentiation [Qian et al., 1997, supra].
Growth factors, such as FGF2, are understood to work directly on hESCs, NSCs, and other stem and progenitor cells, and/or, in some methods, indirectly by stimulating feeder cells in the cell culture to produce this and other growth factors [Bendall, et al., 2007, supra]. However, levels of FGF2 and other growth factors are unstable in these cell cultures, and must be frequently replaced. For example, the half-life of FGF2 is less than 24 hours under conditions typically used to culture stem and progenitor cells. [McKinnon et al., 1990, Neuron. 1990 November; 5(5):603-14]. Consequently, standard methods for maintaining hESC cultures require feeding the cells every day with soluble FGF2 and/or other growth factors in order to maintain effective amounts of active FGF2 polypeptide and/or those other growth factors [Fasano C A, et al. (2010) Cell Stem Cell 6, 336-47]. Despite that laborious, time-consuming process, however, daily feeding of FGF2 and/or other growth factors to stem and progenitor cells still results in (1) significant variation of growth factor levels, with very high levels of FGF2 for the few hours immediately after feeding and very low FGF2 levels present during the few hours prior to the next daily feeding, and (2) limited effectiveness, since hESCs still gradually differentiate, albeit at a slower rate than in the absence of daily feeding with FGF2.
Biodegradeable “microspheres” and “millicylinders” prepared from biocompatible polyesters of glycolic and lactic acids (“PLGA”) are known for delivering protein drugs to patients, and PLGA millicylinders encapsulated with recombinant human FGF2 (also known as “basic fibroblast growth factor” or “bFGF”) have been described by Zhu et al. (Nature Biotechnology (2000) 18:52-57) for such applications. Olaye et al. (European Cells and Materials (2008) 16 (Suppl. 3):86) teach that “PLGA microspheres have been extensively used for the sustained delivery of growth factors for embryonic stem cell differentiation,” and report that PLGA microsphere-based scaffolds were successfully used to deliver certain growth factors—specifically Asc, Dex and TGF-β1—for differentiation of murine embryonic stem cells into osteoblast and chondrocyte-like cells. PVA-based polymer coatings and hydrogen particles for cell culturing have also been described, e.g., by Hemperly et al., U.S. Patent Application Publication No. 2004/0209361 and Keith et al., U.S. Patent Application Publication No. 2004/0209360, respectively. These polymer coatings and particles promote cell adhesion, and may also provide slow release of “bioaffecting molecules,” such as growth factors. Id. More recent publications emphasize a role for FGF2 in promoting cellular differentiation, and discuss hydrogels, microspheres and the like for tissue specific delivery of that growth fact, e.g., to promote tissue regeneration and wound healing. For review, see Yun et al., J. Tissue Eng. (Nov. 7, 2010) 2010:218142; see also Macdonald et al., Biomacromolecules (Aug. 9, 2010) 11(8):2053-2059. Hence, the use of such “sustained release” preparations in stem cell cultures has been limited to delivering growth factors for stem cell differentiation. The sustained release of growth factors, using such compositions or otherwise, for maintaining cells in an undifferentiated state is believed to be heretofore unknown. Hence, there remains a need in the art for improved methods of culturing stem cells and other undifferentiated cells, and for maintaining such cells in an undifferentiated state.